Radiohalogens such as radioactive isotopes of iodine, bromine, fluorine and astatine are useful for a variety of diagnostic and therapeutic applications. In order to enhance the delivery of the diagnostically or therapeutically effective radiohalogen to a desired target site, in vivo or in vitro, the radiohalogen is often coupled to a protein, particularly a glycoprotein such as an antibody or antibody fragment which has specific avidity or affinity for the desired target site. Unfortunately, with the radiohalogenated conjugates developed to date, there has been no method for effectively maintaining the antibody's ability to specifically localize at a target site in vivo and avoid delivery to non-target sites while at the same avoiding dehalogenation of the conjugate with undesirable localization of the free radiohalogen at non-target sites including especially the thyroid.
Direct radiohalogenation of glycoproteins particularly antibodies or antibody fragments has been unsatisfactory because the oxidizing reaction conditions required for direct radiohalogenation are particularly harmful and tend to destroy the immunospecificity of the antibody molecule. Indeed, some antibodies cannot be directly radiohalogenated without substantially destroying the antigen binding ability of the antibody (Powe, 1986, Hybridoma 5 (Suppl. 1):S39-S42). Moreover, even if the antibody activity or ability to bind antigen is maintained to an appreciable extent, directly radiohalogenated antibodies are unsuited for in vivo applications because the halogenation occurs primarily on tyrosinyl residues of the protein moiety of the antibody. The radiohalogenated tyrosinyl residues are particularly unsuitable because of the susceptibility of such structures to dehalogenation enzymes present in vivo such as the deiodinase enzymes which catabolize thyroid hormones such as thyroxine. (See generally, Gershengorn et al., 1980, in The Thyroid Gland, M. DeVisscher, ed., New York, Raven Press, p.81).
In order to avoid the problems associated with direct radiohalogenation of antibody molecules, small molecules such as the Bolten-Hunter reagent, N-succinidyl-3-(4-hydroxyphenyl-propionate and the Wood's reagent, methyl-4-hydroxybenzimidate have been radioiodinated and then coupled to an antibody molecule under comparatively mild coupling conditions. These molecules, however, have a radioiodine moiety "ortho" to a hydroxy group on an aromatic ring (herein called "ortho halogen") and consequently suffer from similar susceptibility to in vivo deiodinase enzymes as do the radioiodinated tyrosine residues of directly radioiodinated antibodies.
European Patent Application No. 86303757.8 published Dec. 3, 1986 describes a method for radiohalogenation of an antibody molecule using an aromatic ring which does not contain a hydroxy functionality and which is substituted in a para or meta position. The method requires the following steps: (1) metalation of a haloaryl compound with an organmetallic group selected from Sn(n-Bu).sub.3 and SnMe.sub.3 ; (2) transmetalation in a site-specific reaction of the resulting aryltin compound by a group selected from HgX.sub.2, Hg(OAc).sub.2, BX.sub.3 or BZ.sub.3 in which X is Cl, Br or I and Z is alkyl or alkoxy; (3) subsequent radiohalogenation of the metalated compound by a demetalation reaction; and (4) addition of a functional group for conjugation to a protein, preferably prior to the radiohalogenation (See also, Wilbur et al., 1986, J. Labelled Compounds and Radiopharmaceuticals XXIII (10-12):1304-06).
Unlike the method and compositions of European Patent Application No. 86303757.8, the compositions of the present invention encompass radiohalogenated aromatic compounds comprising an aromatic ring activated by an hydroxy, alkoxy or hydroxyalkoxy moiety which are formed by a rapid, easy method using starting materials which are more readily available from commercial sources. Additionally, the present method avoids cumbersome metalation of the aromatic moiety. Thus the present method avoids the need for purification steps to remove any residual or unreacted metal such as Hg which could present a problem when the products are administered in vivo. The hydroxy, alkoxy or hydroxyalkoxy moiety may enhance the aqueous solubility of the present radiohalogen intermediates. The present compositions further comprise a reactive amine moiety for site-specific attachment to an oxidized carbohydrate moiety of an antibody which is not part of nor directly involved with the antigen binding region of the antibody molecule. Thus, the present compositions and methods are particularly well suited for in vivo targeted delivery of a radiohalogen moiety that is therapeutically or diagnostically effective. The present methods and compositions are intended and will satisfy a long-felt need for easy, efficient methods and compositions of radiohalogenated products that selectively localize at a target site in vivo; are not localized at non-target sites; and do not readily release radiohalogen when administered in vivo.